ncRNAs-mediated TIMELESS overexpression in lung adenocarcinoma correlates with reduced tumor immune cell infiltration and poor prognosis

Lung adenocarcinoma (LUAD) has a poor prognosis. Circadian genes such as TIMELESS have been associated with several pathologies, including cancer. The expression of TIMELESS and the relationship between TIMELESS, infiltration of tumors and prognosis in LUAD requires further investigation. In this study, we investigated the expression of TIMELESS and its association with survival across several types of human cancer using data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression Program. Noncoding RNAs (ncRNAs) regulating overexpression of TIMELESS in lung adenocarcinoma (LUAD) were explored with expression, correlation, and survival analyses. Immune cell infiltration and biomarkers were analyzed between different TIMELESS expression levels. The relationship between TIMELESS expression and immunophenoscores, which were used to predict response to immunotherapy, was evaluated. TIMELESS was identified as a potential oncogene in LUAD. NcRNA analysis showed MIR4435-2HG/hsa-miR-1-3p may interact with TIMELESS in a competitive endogenous RNA network in LUAD tumor tissues. Most immune cells were significantly decreased in TCGA LUAD tumor tissues with high TIMELESS expression except for CD4+T cells and Th2 cells. TIMELESS expression in LUAD tumor tissues was significantly negatively correlated with neutrophil biomarkers, dendritic cell biomarkers (HLA-DPB1, HLA-DQB1, HLA-DRA, HLA-DPA1, CD1C) and an immunophenoscore that predicted outcomes associated with the use of immune checkpoint inhibitors. These findings imply that ncRNAs-mediated TIMELESS overexpression in LUAD tumor tissues correlated with poor prognosis, reduced immune cell infiltration in the tumor microenvironment, and poor response to immune checkpoint inhibitors.


Introduction
In 2020, lung cancer was responsible for approximately 1.8 million deaths, and was the most common cause of cancer-related mortality [1].Non-small cell lung cancer (NSCLC) is a key type of lung cancer.Lung adenocarcinoma (LUAD) is the most common subtype of NSCLC, and incidence rates have continued to increase in recent years [2].The application of novel diagnostic and therapeutic technologies, including immunotherapy [3,4], has improved prognosis in NSCLC, but the overall five-year survival rate remains low, at 25% according to The Surveillance, Epidemiology, and End Results (SEER) [5].There is clinical need for predictive biomarkers and novel therapeutic strategies in LUAD.
The molecular circadian clock is an endogenous timing system that has been associated with several pathologies, including cancer [6,7].TIMELESS is a core circadian gene that controls circadian rhythmicity.TIMELESS may be involved in carcinogenesis by modulating cell cycles, DNA repair, and tumor immunity [8][9][10][11][12][13].The expression of TIMELESS is increased and associated with poor patient outcomes in NSCLC [14,15].The expression of TIMELESS and the association of TIMELESS with tumor immune cell infiltration and prognosis in LUAD remain to be elucidated.
Competitive endogenous RNAs (ceRNAs), including long non-coding RNAs (lncRNA), pseudogene transcripts, and circular RNAs (circRNAs), regulate each other as they share microRNA (miRNA) recognition elements [16].lncRNAs regulate transcription in mammalian circadian systems [17,18].The objectives of this study were to investigate: 1) the expression of TIMELESS and its association with survival across several types of human cancer; 2) the regulation of TIMELESS by non-coding RNAs (ncRNAs) in LUAD; and 3) the relationship between TIMELESS and infiltration of tumors in LUAD to predict outcomes associated with the use of immune checkpoint inhibitors.Findings indicate that ncRNA mediate upregulation of TIMELESS, which is associated with tumor immune cell infiltration and poor prognosis in LUAD.

Differential genes expression
In the 33 tumor classes included in The Cancer Genome Atlas (TCGA) datasets, we selected 18 tumor types that contained more than five normal tissue samples (Bladder Urothelial Carcinoma BLCA, Breast Invasive Carcinoma BRCA, Cholangiocarcinoma CHOL, Colon Adenocarcinoma COAD, Esophageal Carcinoma ESCA, Glioblastoma Multiforme GBM, Head and Neck Squamous Cell Carcinoma HNSC, Kidney Chromophobe KICH, Kidney Renal Clear Cell Carcinoma KIRC, Kidney Renal Papillary Cell Carcinoma KIRP, Liver Hepatocellular Carcinoma LIHC, Lung Adenocarcinoma LUAD, Lung Squamous Cell Carcinoma LUSC, Prostate Adenocarcinoma PRAD, Rectum adenocarcinoma READ, Stomach Adenocarcinoma STAD, Thyroid Carcinoma THCA, and Uterine Corpus Endometrial Carcinoma UCEC).The mRNA expression data from these 18 cancers were downloaded from TCGA database.Differential expression analysis using the Limma package in R (version 4.0.1)identified altered TIMELESS expression between tumors and adjacent normal tissues.The Gene Expression Profiling Interactive Analysis database (GEPIA) (http://gepia.cancer-pku.cn/)[19] was used to compare and visualize TCGA tumor tissues to normal tissues from TCGA and individuals without cancer from the Genotype Tissue Expression (GTEx) Program.

GEPIA database analysis
Package survival in R was used to determine the overall survival (OS) and disease-free survival (RFS) between different TIMELESS expression and lncRNA expression across multiple cancer types, including LUAD.Two groups of high and low expression were established using the median value as a cut-off.

Tumor immune cell infiltration and response to immune checkpoint inhibitors
Single-sample gene set enrichment analysis (ssGSEA) was used to investigate the proportions of 28 types of immune cells in immune cell infiltration in TCGA LUAD tumor tissues [21].The Cancer Immunome Atlas (TCIA) (https://tcia.at/) was used to predict response to immune checkpoint inhibitors using an immunophenoscore that predicts response to immunotherapy with Cytotoxic T Lymphocyte-Associated Antigen-4 (CTLA-4) and Programmed Death 1(PD-1) blockers [22].Immunophenoscores were correlated with TIMELESS expression using Spearman's correlation.

Statistical analysis
Statistical analyses were performed using online databases or R software, as described above.p <0.05 was considered statistically significant.

TIMELESS expression and prognostic utility
Data from TCGA showed TIMELESS expression in tumor tissues was significantly increased vs. adjacent normal tissues in 16 types of cancer, and significantly decreased vs. adjacent normal tissues in 2 types of cancer (Fig 1A).TIMELESS expression was significantly increased in TCGA tumor tissues across 14 types of cancer vs. normal tissues from individuals without cancer in the GTEx Program (Fig 1B).Among these 14 tumor tissues, high TIMELESS expression in tumor tissues was significantly associated with poor OS in KIRC, LIHC, and LUAD and poor RFS in LIHC and LUAD (Fig 1C).These data suggest TIMELESS may be utilized as a prognostic marker in LUAD.

TIMELESS upstream miRNAs
A total of 85 miRNAs with potential to interact with TIMELESS were identified in TCGA LUAD tumor tissues (Fig 2A).According to published literature, there is negative feedback regulation of the target gene by miRNAs; therefore, we expected a negative correlation between candidate miRNA and TIMELESS expression and downregulation of candidate miR-NAs in LUAD tumor tissues vs. adjacent normal tissues.In TCGA LUAD tumor tissues, TIMELESS expression showed a significant negative correlation with hsa-miR-1-3p, hsa-miR-145-5p, hsa-miR-181a-5p, hsa-miR-224-3p and hsa-miR-551a expression (Table 1

hsa-miR-1-3p upstream lncRNAs
A total of 92 lncRNAs with potential to interact with hsa-miR-1-3p were identified in TCGA LUAD tumor tissues.The lncRNAs most likely to participate in the lncRNA-hsa-miR-1-3p -TIMELESS interaction network in LUAD tumor tissue were selected according to the following criteria: significant upregulation of lncRNA expression in LUAD tumor tissues vs. adjacent normal tissues, significant negative correlation between lncRNA expression and hsa-miR-1-3p expression in LUAD tumor tissues, and significant association of high lncRNA expression in tumor tissues with poor prognosis in LUAD.Among the 92 lncRNAs with potential to interact with hsa-miR-1-3p, only MIR4435-2HG, MIAT, and CYTOR (LINC00152) were significantly upregulated in TCGA LUAD tumor tissues vs. adjacent normal tissues, with expression that significantly negatively correlated with hsa-miR-1-3p expression (Fig 3A and 3B).High MIR4435-2HG expression in tumor tissues was significantly associated with poor OS in the TCGA LUAD cohort (Fig 3C).Overexpressed CYTOR tended towards poor OS in LUAD but did not reach significance.These data suggest MIR4435-2HG and CYTOR have a potential role in the lncRNA-hsa-miR-1-3p -TIMELESS interaction network in LUAD (Fig 3D).

Tumor immune cell infiltration and response to immune checkpoint inhibitors
TIMELESS plays a critical role in the immune system.ssGSEA scores for most immune cells were significantly decreased in TCGA LUAD tumor tissues with high TIMELESS expression vs. low TIMELESS expression.ssGSEA scores for activated CD4 + T cells and the immunosuppressive Th2 cells were significantly increased in TCGA LUAD tumor tissues with high TIME-LESS expression vs. low TIMELESS expression (Fig 4A).A heatmap of immune cell infiltration confirmed that increased TIMELESS expression was associated with CD4 + T and Th2 cell infiltration in TCGA LUAD tumor tissues (Fig 4B).TIMELESS expression was significantly negatively correlated with neutrophil biomarkers (CEACAM8) and dendritic cell biomarkers (HLA-DPB1, HLA-DQB1, HLA-DRA, HLA-DPA1, CD1C) in TCGA LUAD tumor tissues (Table 2).These data suggest TIMELESS is involved in immune regulation of the LUAD tumor microenvironment.TIMELESS expression was significantly negatively correlated with an immunophenoscore that predicted response to immune checkpoint inhibitors in TCGA LUAD tumor tissues (Fig 5).These data suggest patients with LUAD and high TIME-LESS expression in their tumor tissue may be less responsive to immune checkpoint inhibitors than patients with lower TIMELESS expression in their tumor tissue.

Discussion
This study used data from TCGA and the GTEx Program to explore TIMELESS expression and survival outcomes in human cancers.High TIMELESS expression in tumor tissue was significantly associated with poor OS in KIRC, LIHC, and LUAD and poor RFS in LIHC and LUAD.Consistent with this, other studies have shown a correlation between TIMELESS overexpression and poor prognosis in lung cancer [14,15,23], and TIMELESS has been used to build a predictive model of survival in lung cancer [24].However, the regulation and mechanism of action of TIMELESS in lung cancer remains to be elucidated.
ncRNAs, including miRNAs and lncRNAs, are one component of post transcriptional regulation of gene expression.In the present study, TIMELESS expression was significantly negatively correlated with hsa-miR-1-3p expression in LUAD tumor tissues, and high hsa-miR-1- 3p expression was significantly associated with improved OS in LUAD.Previous reports indicate that miR-1-3p has an inhibitory role in lung cancer by targeting CELSR3 and FAM83A and modulating the viability, migration, and invasion of tumor cells [25][26][27].According to the ceRNA hypothesis [28], lncRNAs in the lncRNA-hsa-miR-1-3p -TIMELESS interaction network in LUAD should be oncogenic.Expression, survival and correlation analyses identified MIR4435−2HG as a candidate oncogenic lncRNA in the lncRNA-hsa-miR-1-3p -TIMELESS Research has revealed that some genes including circadian genes can alter tumor immune cell infiltration and influence prognosis [12,[33][34][35].The present study adds to this evidence-   can maintain anti-tumor immunity [36].Further, TIMELESS expression in LUAD tumor tissues was significantly negatively correlated with an immunophenoscore that predicted response to immune checkpoint inhibitors [22], reflecting the impact of TIMELESS on immune cell infiltration.As is a circadian gene, immune checkpoint inhibitor regimens may need to be optimally timed with a patient's intrinsic rhythms to improve outcomes.Currently, only chronoradiotherapy and chronochemotherapy have been investigated across several cancers [37][38][39].This study had some limitations.First, data were extracted from publicly available databases.Second, immunity-related analyses were based on bioinformatics, with no physiological validation.Third, lung cancer is associated with multiple molecular abnormalities, but we only focused on the lncRNA-miRNA-interaction network, potentially excluding other critical genes.

Conclusions
In summary, this study identified TIMELESS as a potential oncogene across multiple cancers, including LUAD.High TIMELESS expression in tumor tissues was significantly associated with poor OS and RFS in LUAD.MIR4435-2HG and hsa-miR-1-3p may interact with TIME-LESS in a ceRNA network in LUAD tumor tissues.TIMELESS might exert its oncogenic role by decreasing immune cell infiltration in the LUAD tumor microenvironment, and TIMELESS expression in LUAD tumor tissues may predict a poor response to immune checkpoint inhibitors.Basic science and clinical trials are required to validate these findings.

Fig 1 .
Fig 1. TIMELESS expression and prognostic value across multiple types of cancer.(A) TIMELESS expression in tumor tissues compared to adjacent normal tissues across 18 types of cancer in TCGA.(B) TIMELESS expression in TCGA tumor tissues (red) across18 types of cancer compared to normal tissues (grey) from TCGA and individuals without cancer from the GTEx Program.(C) The prognostic value of TIMELESS in LUAD (right two figures) and LIHC (left two figures).*p value < 0.05; **p value < 0.01; ***p value < 0.001.https://doi.org/10.1371/journal.pone.0296829.g001